Inhibitory IL-10-producing CD4+ T cells are T-bet-dependent and facilitate cytomegalovirus persistence via coexpression of arginase-1

Inhibitory CD4+ T cells have been linked with suboptimal immune responses against cancer and pathogen chronicity. However, the mechanisms that underpin the development of these regulatory cells, especially in the context of ongoing antigen exposure, have remained obscure. To address this knowledge gap, we undertook a comprehensive functional, phenotypic, and transcriptomic analysis of interleukin (IL)-10-producing CD4+ T cells induced by chronic infection with murine cytomegalovirus (MCMV). We identified these cells as clonally expanded and highly differentiated TH1-like cells that developed in a T-bet-dependent manner and coexpressed arginase-1 (Arg1), which promotes the catalytic breakdown of L-arginine. Mice lacking Arg1-expressing CD4+ T cells exhibited more robust antiviral immunity and were better able to control MCMV. Conditional deletion of T-bet in the CD4+ lineage suppressed the development of these inhibitory cells and also enhanced immune control of MCMV. Collectively, these data elucidated the ontogeny of IL-10-producing CD4+ T cells and revealed a previously unappreciated mechanism of immune regulation, whereby viral persistence was facilitated by the site-specific delivery of Arg1.


Introduction
Immune dysregulation occurs during many persistent viral infections. High levels of ongoing viral replication, which characterize human immunodeficiency virus (HIV), hepatitis B virus (HBV), and, in mice, lymphocytic choriomeningitis virus (LCMV), typically lead to T cell exhaustion, defined by impaired effector functions, the expression of inhibitory cytokines and receptors (Wherry, 2011), and substantial alterations in cellular gene expression (Doering et al., 2012). Moreover, inducible and naturally occurring FoxP3 + regulatory T cells accumulate during many chronic viral infections, presumably to limit excessive immune activation (Veiga-Parga et al., 2013), and T helper (T H )1-like cells that express the immunosuppressive cytokine interleukin (IL)-10 can be induced by LCMV (Parish et al., 2014), HIV (Graziosi et al., 1994), and human/murine cytomegalovirus (HCMV/MCMV) Jones et al., 2010;Mason et al., 2013). Evidence from parasitic infections suggests that IL-10producing T H 1-like cells protect against immune pathology, akin to classical FoxP3 + regulatory T cells (Anderson et al., 2007;Jankovic et al., 2007). However, experimental deletion of IL-10 production in T cells has been shown to promote the clearance of LCMV without any obvious collateral effects Parish et al., 2014;Richter et al., 2013), suggesting a potential therapeutic role for similar manipulations in humans, albeit with the possibility of an attendant risk to the development of CD8 + T cell memory (Laidlaw et al., 2015).
The mechanisms that induce IL-10 expression in T cells require further clarification, despite proposed roles for costimulatory receptors, cytokines, transcription factors, and signals delivered via the T cell receptor (TCR) (Saraiva and O'Garra, 2010). For example, chronic antigen exposure and the transcription factor Blimp-1 appear to be important for the development of IL-10-producing CD4 + T cells in mice infected with LCMV (Parish et al., 2014), and the inhibitory receptor TIGIT is known to act upstream of IL-10 (Schorer et al., 2020). However, it is clear that viral persistence can be facilitated by IL-10, exemplified in the context of MCMV infection by ongoing replication in the salivary glands (SGs) (Humphreys et al., 2007;Mandaric et al., 2012).
Interferon (IFN)γ-expressing CD4 + T cells have been shown to limit viral replication in the SGs of mice infected with MCMV (Jonjić et al., 1989;Lucin et al., 1992;Walton et al., 2011). Nonetheless, CD4 + T cells also represent an important source of IL-10 Humphreys et al., 2007), the production of which is promoted by IL-27 during acute infection with MCMV. In contrast, less is known about the mucosal IL-10-producing CD4 + T cells that appear during chronic infection with MCMV, which are phenotypically distinct from type 1 regulatory T (Tr1) cells, specifically lacking concurrent expression of CD49d and LAG-3, and develop independently of IL-27 . These cells express high levels of various transcription factors, such as c-Maf and T-bet , and often coexpress other molecules with putative inhibitory functions, such as PD-1, TIM-3, and IL-21 (Apetoh et al., 2010;Awasthi et al., 2007;Chihara et al., 2018;Pot et al., 2009;Zhu et al., 2015). However, the functional relevance of these characteristics has remained obscure, along with the ontogeny of IL-10-producing CD4 + T cells during chronic infection with MCMV.
To address these issues, we performed a comprehensive functional, phenotypic, and transcriptomic analysis of IL-10-producing CD4 + T cells isolated from the SGs of mice infected with MCMV. Our data revealed that these cells were clonally expanded and highly differentiated T H 1-like cells with gene expression signatures that indicated a key developmental role for T-bet. In addition, we identified an inhibitory effect attributable to arginase-1 (Arg1), which was upregulated among IL-10producing CD4 + T cells during viral chronicity and facilitated the site-specific persistence of MCMV.

IL-10-producing CD4 + T cells display a T H 1-like profile
To better understand the development and functionality of inhibitory CD4 + T cells that develop during viral chronicity, we infected MCMV IL-10 reporter (10BiT) mice with MCMV. These mice express Thy1.1 under the Il10 promoter (Maynard et al., 2007). Unlike mucosal sites in the respiratory tract (Zhang et al., 2019), ongoing viral replication in this model occurs primarily in the SGs, facilitated by the induction of CD4 + T cells that produce IL-10 (Humphreys et al., 2007), which peak on day 14 post-infection (p.i.) . At this time point, we found that approximately 10-30% of CD4 + T cells in the SGs were Thy1.1 + , of which ~95% displayed an effector memory phenotype (CD44 hi CD62L lo ) (Figure 1-figure supplement 1A). IL-10-producing CD4 + T cells were also induced by polyclonal stimulation and universally expressed Thy1.1 (Figure 1-figure supplement 1B).
Thy1.1 + CD4 + T cells are known to express the T H 1-associated chemokine receptors CXCR3 and CCR5 . We found that MCMV-induced Thy1.1 + CD4 + T cells shared many transcripts with CD4 + T H 1 cells generated in vitro (Stubbington et al., 2015;Figure 1E), encompassing genes associated with numerous cellular and immunological processes (https://doi.org/10.5281/ zenodo.7447477), and further expressed IFNγ in response to polyclonal stimulation at a population frequency of ~25% (Figure 1-figure supplement 1E). These data suggested that Thy1.1 + CD4 + T cells were commonly derived from antigen-specific T H 1 cells, especially given that IFNγ detection via flow cytometry likely underestimates the composite frequency of CD4 + T cells that specifically recognize MCMV (Jeitziner et al., 2013). However, genes associated with the induction of IFNγ, including Il18r1 and il18rap, were actually downregulated in Thy1.1 + CD4 + T cells ( Figure 1A, C, D), and in two of three replicates, a similar pattern was observed for Ifng (https://doi.org/10.5281/zenodo.7243956). Comparable findings were reported previously in functional studies of IFNγ expression at the protein level among IL-10-producing CD4 + T cells specific for HCMV or MCMV Mason et al., 2013).
IL-10 production among CD4 + T cells has been associated with the expression of inhibitory molecules and markers of exhaustion (Saraiva and O'Garra, 2010). Counterintuitively, we found that MCMV-induced Thy1.1 + CD4 + T cells downregulated the exhaustion-associated transcription factor Tox1 but nonetheless expressed a module of inhibitory genes, including Lag3, Fgl2, Havcr2, and Entpd1 ( Figure 1D). These inhibitory molecules were also expressed at the protein level, alongside PD-1 ( Figure 1G and Figure 1-figure supplement 1F). In addition, differential bystander activation seemed unlikely, because Thy1.1 + CD4 + T cells expressed LAG-3 and PD-1 more commonly than Thy1.1 − CD4 + T cells after preselection based on the induction of IFNγ (Figure 1-figure supplement  1E).   Collectively, these data showed that IL-10-producing CD4 + T cells exhibited a highly differentiated T H 1-like profile, characterized by the upregulation of various inhibitory molecules and the downmodulation of IFNγ expression lacking concordance with known signatures of exhaustion, during chronic infection with MCMV.
IL-10-producing CD4 + T cells exhibit prominent clonal structures IL-10-producing CD4 + T cells recognize a broad range of viral antigens during chronic infection with MCMV . To characterize these interactions in more detail and evaluate the clonal relationship between IL-10 + (Thy1.1 + ) and IL-10 − (Thy1.1 − ) CD4 + T cells, we used a next-generation approach to sequence the corresponding TCRs.
It should be noted that none of these differences were absolute. For example, the clusters of TCRβ variants identified among Thy1.1 + CD4 + T cells also occurred at lower cumulative frequencies among Thy1.1 − CD4 + T cells ( Figure 2E-G), and the TCRα and TCRβ repertoires overlapped considerably between Thy1.1 + CD4 + T cells and Thy1.1 − CD4 + T cells ( Figure 2-figure supplement 1B). Moreover, there were no prominent differences in the physicochemical properties of amino acids in the central parts of the CDR3α and CDR3β loops, which generally differ among functionally discrete subsets of CD4 + T cells (Kasatskaya et al., 2020), to indicate an ontogenetic divergence between Thy1.1 + CD4 + T cells and Thy1.1 − CD4 + T cells (Figure 2-figure supplement 1C).
Collectively, these data revealed the presence of common molecular signatures that predominated among Thy1.1 + CD4 + T cells, consistent with the notion of an antigen-driven process of differentiation leading to the production of IL-10.

IL-10-producing CD4 + T cells are enriched for expression of Arg1
Our analysis of inhibitory gene expression revealed one particularly intriguing feature, namely that Thy1.1 + CD4 + T cells significantly upregulated Arg1 ( Figure 1D). Arg1 promotes the catalytic breakdown of L-arginine (Munder, 2009) and has been shown to inhibit the proliferation of T cells (Czystowska-Kuzmicz et al., 2019;Rodriguez et al., 2004;Rodriguez et al., 2002). A previous study also reported that T cells could express Arg1 (Washburn et al., 2019), although the functional relevance of this observation has remained obscure.
To address this knowledge gap, we first confirmed expression at the protein level via Western blotting ( Figure 3A) in experiments incorporating control mice lacking the ability to express Arg1 in the CD4 + lineage (Cd4 Cre/+ Arg1 flox/flox ). We then revealed the open chromatin structure around Arg1 in Thy1.1 + CD4 + T cells ( Figure 3B) and further probed the expression of Arg1 versus Thy1.1 among cytometry plots showing the expression of CD39 and TIM-3 among Thy1.1 + CD4 + T cells (red) and Thy1.1 − CD4 + T cells (blue). Data are shown as pooled analyses from a minimum of n = 10 mice per group representing two independent experiments.
The online version of this article includes the following source data and figure supplement(s) for figure 1: Source data 1. Interleukin (IL)-10-producing CD4 + T cells display a T H 1-like profile.

Figure supplement 1. Characterization of CD4 + T cells isolated from the salivary glands (SGs) after infection with murine cytomegalovirus (MCMV).
Figure supplement 1-source data 1. Characterization of CD4 + T cells isolated from the salivary glands (SGs) after infection with murine cytomegalovirus (MCMV).

Figure 1 continued
CD4 + T cells via flow cytometry ( Figure 3C). Our data showed that Arg1 was expressed by CD4 + T cells almost exclusively in the SGs ( Figure 3A, C). Of note, Arg1 expression was also detected among CD8 + T cells but not among NK T cells via flow cytometry, although intracellular discrimination was subtle ( Figure 3-figure supplement 1A). Depletion experiments nonetheless revealed that only CD4 + T group 1 group 2 group 3 group 1 group 2 group 3  IL-10-producing T H 1 cells can be induced experimentally via high-dose antigen stimulation in the presence of IL-12 (Saraiva et al., 2009). Accordingly, we hypothesized that Arg1 + IL-10-producing CD4 + T cells might develop under similar conditions in vitro, given the corresponding T H 1-like profile observed during chronic infection with MCMV. To test this notion, we stimulated ovalbumin  The online version of this article includes the following source data and figure supplement(s) for figure 3: Source data 1. Interleukin (IL)-10-producing CD4 + T cells are enriched for expression of arginase-1 (Arg1).

CD4 + T cells promote viral persistence via expression of Arg1
To explore the biological relevance of our findings, we infected Cd4 +/+ Arg1 flox/flox and Cd4 Cre/+ Arg1 flox/ flox mice with MCMV. Lineage-specific deletion of Arg1 did not impact the function or phenotype of CD4 + T cells in naive mice (Figure 3-figure supplement 1C, D). Higher numbers of IFNγ-expressing CD4 + T cells ( Figure 4A) and higher frequencies of proliferating (Ki-67 + ) CD4 + T cells ( Figure 4B) were nonetheless observed after viral antigen stimulation in the SGs of Cd4 Cre/+ Arg1 flox/flox versus Cd4 +/+ Arg-1 flox/flox mice during the chronic phase of infection with MCMV. These data suggested that Arg1 inhibited the proliferation of CD4 + T cells in vivo, consistent with a previous in vitro study (Munder et al., 2006). Similarly, higher numbers of virus-specific CD8 + T cells, quantified using tetrameric antigen probes, were detected in the spleens of Cd4 Cre/+ Arg1 flox/flox versus Cd4 +/+ Arg1 flox/flox mice on day 30 p.i. ( Figure 4C). In contrast, Arg1 deletion had no significant impact on the development of virus-specific IL-10-producing CD4 + T cells in the SGs ( Figure 4D).
IFNγ-expressing CD4 + T cells are known to restrict MCMV replication in the SGs (Walton et al., 2011). Accordingly, we found that viral shedding in the saliva ( Figure 4E) and viral replication in the SGs ( Figure 4F) were reduced in Cd4 Cre/+ Arg1 flox/flox versus Cd4 +/+ Arg1 flox/flox mice during the chronic phase of infection with MCMV. Importantly, no differences in viral replication were observed between Cd4 Cre/+ Arg1 flox/flox and control mice at an earlier time point (day 14 p.i.) (Figure 4-figure supplement  1A), and deletion of Arg1 in myeloid cells, achieved using Lyz2 Cre/+ Arg1 flox/flox mice, had no impact on viral shedding in the saliva or viral replication in the SGs (Figure 4-figure supplement 1B, C). Of note, there was also no evidence that Cd4 Cre/+ Arg1 flox/flox mice were more susceptible to virusinduced autoimmunity, as indicated by anti-Sjögrens syndrome antigen (SSA) titers comparable to those observed in Cd4 +/+ Arg1 flox/flox mice (Figure 4-figure supplement 1D).
Collectively, these data indicated that Arg1 expression by CD4 + T cells selectively inhibited the accumulation of virus-specific CD4 + and CD8 + T cells in the SGs, leading to suboptimal immune control of viral replication during chronic infection with MCMV.

IL-10-producing CD4 + T cells develop in a T-bet-dependent manner
In line with our previous work , we noted that Thy1.1 + CD4 + T cells expressed higher amounts of T-bet at the protein level than Thy1.1 − CD4 + T cells ( Figure 5A), and concordantly, we found that open chromatin was enriched in the Tbx21 region of Thy1.1 + CD4 + T cells versus Thy1.1 − CD4 + T cells ( Figure 5B). No such differences in expression intensity were observed for the related T-box transcription factor Eomesodermin (Eomes) (Figure 5-figure supplement 1A,  B), which promotes the development of Tr1 cells (Roessner et al., 2021;Zhang et al., 2017). We also detected considerable overlap between the gene expression profiles of Thy1.1 + CD4 + T cells and T-bet-orchestrated CD4 + T H 1 cells generated in vitro (Zhu et al., 2012; Figure 5C). Moreover, T-bet suppresses the expression of TCF1/7 and IL-7R (Dominguez et al., 2015;Oestreich et al., 2011), mimicking key phenotypic features of Thy1.1 + CD4 + T cells ( Figure 1D, F). On the basis of these observations, we hypothesized that T-bet promoted the development of Arg1 + IL-10-producing CD4 + T cells during chronic infection with MCMV.
To test this notion, we crossed tamoxifen-inducible Cd4 CreERT2/+ mice with Tbx21 flox/flox mice (Intlekofer et al., 2008), allowing us to suppress T-bet expression at the onset of viral chronicity (day 7 p.i.). These mice were further crossed to incorporate a Rosa26 flSTOPtdRFP allele (Luche et al., 2007), enabling the identification of cells in which Cre was expressed via the detection of tandem-dimer red fluorescent protein (tdRFP). Mice were then infected with MCMV. Tamoxifen was administered daily for 5 days from day 7 p.i. to deplete T-bet after initial antigen exposure in a CD4-dependent manner, an   effect that clearly associated with the coincident expression of RFP ( Figure 5D and Figure 5-figure  supplement 1C).
CD4-specific T-bet depletion reduced the accumulation of virus-specific IL-10-producing CD4 + T cells ( Figure 6A) and Arg1 + CD4 + T cells by day 14 p.i. (Figure 6B, C). However, it seemed unlikely that T-bet directly stimulated the expression of IL-10 and Arg1, because the corresponding binding motifs were not preferentially accessible in the Il10 and Arg1 regions ( Figure 1B and Figure 3B). Instead, we observed a shift toward a less differentiated phenotype in Cd4 CreERT2/+ Tbx21 flox/flox mice ( Figure 6D-G),

cells develop in a T-bet-dependent manner. (A-J)
Cd4 +/+ Tbx21 flox/flox (Cd4 +/+ ) and Cd4 CreERT2/+ Tbx21 flox/ flox (Cd4 Cre/+ ) mice were infected with 3 × 10 4 pfu of murine cytomegalovirus (MCMV). Tamoxifen was administered (+Tam) or withheld (−Tam) from days 7 to 12 p.i. Leukocytes were isolated from the salivary glands (SGs) on day 14 p.i. (A) MCMV-specific CD4 + T cell responses in the SGs measured using flow cytometry to detect IL-10. Immunodominant peptides were pooled for stimulation. Data are shown as mean ± standard error of the mean (SEM; n = 4-5 mice per group representing two independent experiments). **p < 0.01 (Mann-Whitney U test). Summary bar graph (B) and representative histograms (C) showing the expression of arginase-1 (Arg1) among CD4 + T cells measured via flow cytometry. Data are shown as mean ± SEM (n = 5 mice per group representing two independent experiments). ***p < 0.001 (Mann-Whitney U test). Summary bar graphs (left) and representative histograms (right) showing the expression of CD44 (D), PD-1 (E), CD62L (F), and IL-7R (G) among CD4 + T cells measured via flow cytometry. Data in (D-F) are shown as mean ± SEM (n = 5 mice per group representing two independent experiments). Data in (G) are shown as mean ± SEM (n = 3-4 mice per group representing two independent experiments). *p < 0.05, **p < 0.01 (Mann-Whitney U test). (H) MCMV-specific CD4 + T cell responses in the SGs measured using flow cytometry to detect interferon (IFN)γ. Immunodominant peptides were pooled for stimulation. Data are shown as mean ± SEM (n = 4-5 mice per group representing two independent experiments). *p < 0.05 (Mann-Whitney U test). (I) MCMV replication in SG homogenates on day 14 p.i. measured via plaque assay. Data are shown as individual points with median values (n = 10-14 mice per group pooled from three independent experiments). **p < 0.01 (Mann-Whitney U test). (J) Viral genomes in saliva on days 7, 14, 21, and 28 p.i. measured via qPCR. Data are shown as mean Figure 6 continued on next page with decreased expression of CD44 ( Figure 6D) and PD-1 ( Figure 6E) and increased expression of CD62L ( Figure 6F) and IL-7R ( Figure 6G) in the absence of T-bet. In addition, T-bet depletion was associated with increased numbers of virus-specific IFNγ-expressing CD4 + T cells ( Figure 6H) and enhanced control of viral replication in the SGs ( Figure 6I), as well as reduced salivary shedding of MCMV ( Figure 6J). In accordance with the observation that IL-10-producing CD4 + T cells occur transiently during the early stages of viral chronicity , we also found that CD4specific T-bet depletion was no longer protective by day 28 p.i. (Figure 6J and Figure 5-figure  supplement 1D).
Collectively, these results suggested that T-bet exhibited dual functionality under conditions of recurrent antigen stimulation, concurrently driving the accumulation of virus-specific T H 1 responses and promoting the development of Arg1 + IL-10-producing CD4 + T cells, which together shaped immune control of viral replication during the early stages of chronic infection with MCMV.

Discussion
In this study, we identified IL-10-producing CD4 + T cells as highly differentiated T H 1-like cells, which occurred transiently at mucosal sites of viral persistence under the strict governance of T-bet. These cells also expressed the metabolic enzyme Arg1. Further investigation revealed that Arg1 expression was a critical immune regulatory function of CD4 + T cells, suppressing antiviral immunity and facilitating viral chronicity in the context of infection with MCMV.
Our finding that Arg1 was one of the putative immune regulatory genes expressed by IL-10producing CD4 + T cells was initially counterintuitive, given the reported lack of Arg1 protein expression by human CD4 + T cells in vitro (Geiger et al., 2016). However, Arg1 expression by CD4 + and CD8 + T cells has been described in patients with sepsis (Washburn et al., 2019), indicating a potential role for generic infectious stimuli and/or an antigen-driven process mediated via the TCR. The latter possibility is consistent with our repertoire data, which revealed that prominent clonal expansions were associated with the production of IL-10. Importantly, we demonstrated that Arg1 was expressed at the protein level, both in secreted form and intracellularly. It should be noted that relatively weak intracellular signals were detected in flow cytometric analyses of C57BL/6 mice, akin to an earlier study of patients infected with HBV (Pallett et al., 2015). Western blotting experiments nonetheless clearly showed that CD4 + T cells expressed Arg1 in the SGs of mice chronically infected with MCMV. Our flow cytometry experiments also suggested that CD8 + T cells could express Arg1, albeit with the caveat of suboptimal visualization, but importantly, CD4 + T cells secreted far greater amounts of soluble Arg1, at least in the milieu of the SGs.
L-arginine deficiency results in cell cycle arrest via the downregulation of CD3ζ, a key component of the TCR (Rodriguez et al., 2002), and T cell proliferation is impaired in the absence of L-arginine (Rodriguez et al., 2007). In line with these observations, we found that improved control of viral replication in mice lacking Arg1 + CD4 + T cells was associated with increased numbers of MCMVspecific effector CD4 + T cells, which are known to limit viral replication in the SGs (Lucin et al., 1992;Walton et al., 2011). Accordingly, the acquisition of Arg1 expression by highly differentiated CD4 + T cells impinges on antiviral immunity, representing an inhibitory mechanism that operates alongside the production of IL-10 Humphreys et al., 2007). It is also notable here that regulatory T cells may act similarly via the expression of Arg2 (Lowe et al., 2019).
IL-10 is regulated by a number of transcription factors, including those required for T cell differentiation, and is induced downstream of the TCR (Saraiva et al., 2020). Our discovery that T-bet promotes the development of IL-10-producing CD4 + T cells aligns with the concept that chronic antigen stimulation can trigger a negative feedback loop via these mechanisms, which concurrently drive activation and differentiation. It nonetheless remains less clear to what extent T-bet directly orchestrates the expression of other inhibitory molecules by CD4 + T cells. There was no notable ± SEM (n = 5 mice per group representing two independent experiments). **p < 0.01 (Mann-Whitney U test). Data in (A-I) show all groups after the administration of tamoxifen.
The online version of this article includes the following source data for figure 6: Source data 1. Interleukin (IL)-10-producing CD4 + T cells develop in a T-bet-dependent manner.

Figure 6 continued
increase in chromatin-accessible binding motifs for T-bet within either the Il10 or Arg1 genes in IL-10producing CD4 + T cells. However, we did find that deletion of T-bet after acute infection limited the accumulation of highly differentiated CD4 + T cells, including those expressing Arg1 and IL-10. In line with this observation, which suggested a differentiation-linked process of functional remodeling driven by chronic antigen exposure, T-bet is known to promote the differentiation of CD8 + T cells (Dominguez et al., 2015;Omilusik et al., 2015) and inhibit the expression of TCF1/7 (Omilusik et al., 2015) and IL-7R (Intlekofer et al., 2007), both of which were downregulated among Arg1 + IL-10producing CD4 + T cells induced by MCMV.
The mechanisms that directly induce the expression of Il10 and Arg1 in CD4 + T cells remain obscure. Although IL-10-producing CD4 + T cells were characterized by prominent clonal expansions, repertoire analysis revealed no obvious determinative role for the TCR. However, the transcription factor c-Maf, which promotes the expression of IL-10 in multiple T H cell subsets (Gabryšová et al., 2018), is known to be upregulated in IL-10-producing CD4 + T cells during chronic infection with MCMV . We also demonstrated previously that ICOS signaling promotes the accumulation of IL-10-producing CD4 + T cells under the same conditions , and c-Maf acts downstream of ICOS (Bauquet et al., 2009;Nurieva et al., 2003). It therefore seems likely that an ICOS-c-Maf axis participates in the direct induction of Il10, although it is less clear how this applies to Arg1. Moreover, we found that Arg1 + IL-10-producing CD4 + T cells developed optimally in vitro in response to high-dose antigen stimulation in the presence of IL-12, suggesting determinative roles for ERK MAP kinase and the transcription factor STAT4 (Saraiva et al., 2009).
The evolutionary advantage of a mechanism that leads to the production of immune regulatory molecules and consequently facilitates viral persistence without impacting autoimmunity is difficult to explain. One possibility is that some of these molecules, potentially including Arg1, serve to maintain tissue health in the presence of ongoing inflammation. It would be informative in this context to evaluate how Arg1 delivery via CD4 + T cells impacts mucosal pathology over time at sites beyond the SGs. Alternatively, local immune suppression may somehow limit the emergence of more pathogenic viral strains under conditions of persistent replication, thereby protecting the host and the wider population. Irrespective of the precise explanation, it seems clear that viral persistence can be facilitated by this intrinsic regulatory mechanism, a phenomenon that could feasibly extend to pathogens other than MCMV.
In summary, we have demonstrated that T-bet activity during a chronic viral infection can impede antiviral immune control by driving the development of highly differentiated T H 1-like cells that express genes encoding inhibitory molecules, including IL-10 and Arg1. Importantly, these Arg1 + IL-10-producing CD4 + T cells developed in vitro under conditions of extreme antigen stimulation, especially in the presence of IL-12, and the expression of Arg1, which was also secreted in vivo as a soluble immune regulatory protein, facilitated viral replication during the chronic phase of infection with MCMV. These observations are conceivably important not only from a biological perspective but also from a translational perspective, revealing a previously unappreciated mechanism through which CD4 + T cells can suppress potentially harmful immune responses via the regulation of L-arginine.

TCR-seq
Unique molecular identifier (UMI)-labeled 5′-RACE TCR cDNA libraries were synthesized using a Mouse TCR Profiling Kit (MiLaboratories). Indexed samples were pooled and sequenced using a MiSeq System (Illumina). Paired-end reads (150 bp) were extracted and clustered by UMI using MiGEC (Shugay et al., 2014). Sequences were discarded from the analysis if the read count was <4 per cDNA. Error correction was performed using MiGEC. Repertoires were extracted using MiXCR . The weighted average use of bulky, charged, and strongly interacting (aromatic and hydrophobic) amino acids positioned centrally in the CDR3α/β sequences and Trbv gene use (weighted by frequency) were calculated using VDJtools . Diversity was calculated by downsampling the repertoires to an equal number of UMIs (n = 4300 for TCRα and n = 3900 for TCRβ) three separate times and plotting the mean Chao1 index, reflecting lower bound total diversity, and (1 − normalized Shannon-Wiener index), reflecting clonality. Visualization was achieved using PlotQuantileStats in VDJtools. The mean number of unique nucleotide sequences encoding the same amino acid sequence (convergence) was calculated for the 2000 most prevalent clonotypes in each sample using VDJtools. Overlap was calculated for the 2000 most prevalent clonotypes using F2 metrics to estimate sharing at the amino acid level among V gene-matched sequences in each sample. Cluster analysis was performed using the 500 most prevalent clonotypes in pooled samples (Thy1.1 + versus Thy1.1 − ). These datasets were analyzed using tcrgrapher (https://github.com/KseniaMIPT/tcrgrapher), an R library based on ALICE (Pogorelyy et al., 2019). The real and expected numbers of neighbors were calculated for each clonotype with a maximum of n = 1 amino acid mismatch. Clonotypes with a significantly higher number of neighbors than expected on statistical grounds (adjusted p < 0.0001 with Benjamini-Hochberg correction) were identified as tcrgrapher hits. The expected number of neighbors was estimated via generation probabilities calculated for each clonotype using OLGA (Sethna et al., 2019), with the selection factor set at Q = 6.27 (Elhanati et al., 2018).

Quantification of MCMV
Infectious virus was quantified in organs via plaque assay as described previously (Stack et al., 2015). 3T3 cells were purchased directly from the American Type Culture Collection. Mycoplasma infection was excluded prior to assay. Viral loads in oral lavage were quantified via qPCR Kamimura and Lanier, 2014).

Western blotting
Leukocytes were isolated from SGs and spleens as described previously . CD4 + T cells were purified via magnetic separation using a MagniSort Mouse CD4 Positive Selection Kit Quantification of anti-SSA Plasma samples were obtained from cardiac punctures and assessed for anti-SSA/Ro 60 IgG levels via ELISA (Alpha Diagnostics).

Statistics
Sample sizes for next-generation sequencing experiments were calculated using G*Power (https:// www.psychologie.hhu.de/arbeitsgruppen/allgemeine-psychologie-und-arbeitspsychologie/gpower. html), where a minimum of n = 5 pooled mice per group was used to detect a difference in means with 90% power and an α value set at 0.05 across a minimum of three replicates. All outliers were included in the final datasets. Comparisons between two groups were performed using the Mann-Whitney U test. Significance across all tests is reported as *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001. ship (Cardiff University). VVK received infrastructure support from the Deutsche Forschungsgemeinschaft (DFG) Cluster of Excellence "Precision Medicine in Chronic Inflammation" (PMI). OVB, DMC, and DAP were supported by a Ministry of Science and Higher Education of the Russian Federation Subsidy Grant (075-15-2019Grant (075-15- -1789